This invention relates to a high unit, novel alkaline protease and a very advantageous manufacture of the same by cultivation of a novel microorganism producing alkaline protease in an alkaline culture medium containing a carbonate or a culture medium not containing sugars.
Various methods for the manufacture of protease, a protein-decomposing enzyme, having optimal pH in the alkaline side, are known, but methods for the production of a large quantity of alkaline protease using a microorganism and the special media and culture conditions as in the present invention have not been described in the literature.
The present invention is characterized in the microorganism used and its culture conditions for production of a large quantity of novel alkaline protease.
The microorganisms used in the present invention show good growth under culture conditions to be described in detail below, and produce the alkaline protease of the present invention. They are new strains belonging to the Bacillus genus, Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76. The said Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76 have been discovered by the present inventors.
Each of the said strains, Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76 which are employed in the process of the invention, is isolated from the soil collected in the Yamato-machi district of Kitaadachi-gun, Saitama Prefecture, Japan.
The isolation of the microorganisms, Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76 was carried out by a procedure described hereinbelow.
The soil from the district mentioned above was suspended in sterilized water and plated on the following medium:
______________________________________ (Medium composition) ______________________________________ a) Soluble starch 20 g K.sub.2 HPO.sub.4 1 g Yeast extract 5 g Peptone 5 g MgSO.sub.4.7H.sub.2 O 0.2 g Agar 20 g Water 900 ml b) Na.sub.2 CO.sub.3 10 g Water 100 ml (After sterilization at 115.degree. C for 15 min., a) and b) were mixed.) ______________________________________
The plate was incubated at 37.degree. C for 24 to 48 hours.
Thus, a colony of a microorganism which produced a large amount of the said alkaline protease was isolated from colonies on the plate.
This microorganism was called "Bacillus sp. No. 221." The said Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76 were also isolated by the same method.
The strains identified as said Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76 have been deposited with the American Type Culture Collection (ATCC) 12301 Parklawn Drive, Rockville, Maryland, as ATCC access numbers 21522, 21536 and 21537, respectively, and are on deposit with ATCC as unrestricted deposits permitting the public full access to the cultures. The strains are released for distribution to the public as of Apr. 22, 1970, May 5, 1970 and May 5, 1970, respectively. The present inventors found that the said microorganisms, Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76 produce and accumulate a high unit of novel alkaline protease under the culture conditions which will be described hereinbelow, and succeeded in establishing the process for the manufacture of the alkaline protease of the present invention. This alkaline protease is very useful as an additive for detergents.
The said Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76 have the following properties: The microbiological properties were tested by the methods described in "Aerobic Spore-forming Bacteria" by Nathan R. Smith, R. E. Gordon and F. E. Clark (United States Department of Agriculture, Nov. 1952) and "Bergey's Manual of Determinative Bacteriology" (1957).
______________________________________ Bacillus sp. No. 221 (a) Growth in Medium pH in the medium Medium pH 7 pH 10.2* ______________________________________ (1) Bouillon Growth Growth (2) Bouillon-agar " Good growth (3) Glucose-bouillon Poor growth Growth abandant (4) Glucose-bouillon Growth scant Growth abandant agar (5) Gelatin medium -- Good growth (6) Peptone water Growth Growth not good (7) Potato medium Growth Good growth ______________________________________ *1% of Na.sub.2 CO.sub.3 was added
The size of the microorganism is 0.6 - 0.8.mu. .times. 2.0 - 3.0.mu.; the sporangium is slightly swollen and the spore is oval, 0.8 - 1.0.mu. .times. 1.3.mu..
The microorganism has pertrichous flagella, as will be seen in the electron micrograph attached as FIG. 1. The Bacillus grows very well on the medium (glucose, yeast extract, peptone, K.sub.2 HPO.sub.4, MgSO.sub.4 7H.sub.2 O and Na.sub.2 CO.sub.3, pH 10.2) to be described below, as a white colony. The characteristic of this Bacillus is that it grows well in an alkaline medium rather than in a neutral medium in which no sugar is contained. b. Physiological Properties
1. Optimal Growth Conditions: pH 8-10 Temperature: 37.degree. - 40.degree. C Aerobic PA1 2. Conditions under which the bacteria can grow: pH 7-11 Temperature: up to 55.degree. C Aerobic PA1 3. gram stainability: Positive (changeable) PA1 4. Voges-Proskauer reaction: Negative PA1 5. Nitrate is reduced. PA1 6. Catalase: Positive PA1 7. Hydrolysis of gelatine and casein: Positive PA1 8. Hydrolysis of starch: Weak PA1 9. Utilization of citrate: Utilized PA1 10. Utilization of ammonium salts: Utilized PA1 11. Growth detected in 5% sodium chloride solution. PA1 12. Growth detected on glucose-nitrate medium. PA1 13. No growth under anaerobic conditions. PA1 14. No production of gas in nitrate medium under anaerobic conditions. PA1 15. Growth detected in a glucose-asparagine medium. PA1 1. Optimal Growth Conditions: pH 8-10 Temperature: 37.degree. - 40.degree. C Aerobic PA1 2. Conditions under which the bacteria can grow: pH 6.0 - 11 Temperature: up to 56.degree. C Aerobic PA1 3. gram stainability: Positive (changeable) PA1 4. Voges-Proskauer reaction: .+-. PA1 5. Nitrate is reduced. PA1 6. Catalase: Positive PA1 7. Hydrolysis of gelatine and casein: Positive PA1 8. Hydrolysis of starch: Weak PA1 9. Utilization of citrate: Utilized PA1 10. Utilization of ammonium salts: Utilized PA1 11. Poor growth detected in 7% sodium chloride solution. PA1 12. Growth scanty in a glucose-nitrate medium. PA1 13. No growth under anaerobic conditions. PA1 14. No production of gas in nitrate medium under anaerobic conditions. PA1 15. Growth detected in a glucose-asparagine medium. PA1 1. Optimal Growth Conditions: pH 8 - 10 Temperature: 37.degree. - 40.degree. C Aerobic PA1 2. Conditions under which the bacteria can grow: pH 6.0-11 Temperature: up to 56.degree. C Aerobic PA1 3. gram stainability: Positive (changeable) PA1 4. Voges-Proskauer reaction: .+-. PA1 5. Nitrate is reduced. PA1 6. Catalase: Positive PA1 7. Hydrolysis of gelatine and casein: Positive PA1 8. Hydrolysis of startch: Positive PA1 9. Utilization of citrate: Utilized PA1 10. Utilization of ammonium salts: Utilized PA1 11. Growth detected in 7% sodium chloride solution. PA1 12. Growth detected on glucose-nitrate medium. PA1 13. No growth under anaerobic conditions. PA1 14. No production of gas in nitrate medium under anaerobic conditions. PA1 15. Growth detected on glucose-asparagine medium. PA1 i. Sedimentation constant from ultracentrifugation is about 3.3S. PA1 ii. Molecular weight determined using Sephadex (the word "Sephadex" is a registered trade mark.) was a value between 20,000 and 40,000. PA1 iii. The value presumed from amino acid analysis is integral multiple of ca. 16,000. PA1 iv. Molecular weight calculated by the Archibald method is ca. 33,000.
The following experiments were carried out using the medium containing 1% Na.sub.2 CO.sub.3.
c. Utilization of Carbon Source PA0 b. Physiological Properties PA0 c. Utilization of Carbon Source PA0 b. Physiological Properties PA0 c. Utilization of Carbon Source PA0 1. Analytical values: Found: C, 48.04; H, 6.62; N, 16.07; S*, 0.31% * Value calculated from amino acid analysis, which showed that only methionine is present as sulfurcontaining amino acid, and neither cysteine nor cystine are detected. PA0 2. Molecular weight: The following experiments suggest that the molecular weight of this protease is around 20,000 - 30,000. PA0 3. Optimal pH and range of stable pH: As will be clear from FIG. 4, the optimal pH lies between 11 and 12. PA0 4. Assay of Enzyme Activity: PA0 5. range of Working Temperature: PA0 6. conditions for Inactivation by Temperature: PA0 7. inhibition, Activation, and Stabilization: PA0 8. method of Purification: PA0 9. Crystal Structure: PA0 10. electrophoresis:
The microorganism utilizes glucose, mannose, salicin, cellobiose, lactose, sucrose, arabinose, mannitol, and xylose in a medium containing 1% carbonate but production of acid is not clear due to the presence of a large quantity of carbonate.
______________________________________ Bacillus sp. No. 0-4 (a) Growth in Medium pH in the medium Medium pH 7 pH 10.2* ______________________________________ (1) Bouillon Poor growth Growth (2) Bouillon-agar " Good growth (3) Glucose-bouillon " " (4) Glucose-bouillon Growth " agar (5) Gelatin medium -- " (6) Peptone water -- Growth not good (7) Potato medium Growth Good growth ______________________________________ *1% of Na.sub.2 CO.sub.3 was added
The size of the microorganism is 0.5 - 0.7.mu. .times. 2.0 - 3.0.mu.; the sporangium is slightly swollen and the spore is oval, 0.7 - 1.0.mu. .times. 1.2 - 1.3.mu..
The microorganism has pertrichous flagella, as will be seen in the electron micrograph attached as FIG. 2. The said Bacillus grows very well in the medium (soluble starch, yeast extract, peptone, K.sub.2 HPO.sub.4, MgSO.sub.4 7H.sub.2 O and Na.sub.2 CO.sub.3, adjusted at pH 10.2) to be described below as a white colony. The characteristic of this Bacillus is that it grows well in an alkaline medium rather than in a neutral medium.
The following experiments were carried out using the medium containing 1% Na.sub.2 CO.sub.3.
The microorganism utilizes glucose, mannose, salicin, cellobiose, lactose, sucrose, arabinose, mannitol, and xylose in a medium containing 1% carbonate but production of acid is not clear due to the presence of a large quantity of carbonate.
______________________________________ Bacillus sp. No. Y-76 (a) Growth in Medium pH in the medium Medium pH 7 pH 10.2* ______________________________________ (1) Bouillon Growth Growth (2) Bouillon-agar " " (3) Glucose-bouillon " Uniform turbidity, good growth (4) Glucose-bouillon agar " Good growth (5) Gelatin medium -- Good growth liquified (6) Peptone water -- Growth not good (7) Potato medium Growth Good growth ______________________________________ *1% of Na.sub.2 CO.sub.3 was added
The size of the microorganism is 0.5 - 0.7.mu. .times. 2.0 - 3.0.mu.; the sporangium is slightly swollen and the spore is oval, 0.7 - 1.0.mu. .times. 1.2 - 1.3.mu..
The microorganism has pertrichous flagella, as will be seen in the electron micrograph attached as FIG. 3. The said Bacillus grows very well in the medium (soluble starch, yeast extract, peptone, K.sub.2 HPO.sub.4, MgSO.sub.4 7H.sub.2 O and Na.sub.2 CO.sub.3, adjusted at pH 10.2) to be described below, as a white colony. The characteristic of this Bacillus is that it grows well in an alkaline medium rather than neutral medium.
The following experiments were carried out using the medium containing 1% Na.sub.2 CO.sub.3.
The microorganism utilizes glucose, mannose, salicin, cellobiose, lactose, sucrose, arabinose, mannitol, and xylose in a medium containing 1% carbonate but production of acid is not clear due to the presence of a large quantity of carbonate.
Next, the said microorganisms, Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76 are different from each other, and their characteristic different points are shown in the following table:
______________________________________ Microorganisms Bacillus Bacillus Bacillus Properties sp. No. 221 sp. No. 0-4 sp. No. Y-76 ______________________________________ Growth in Very poor Poor growth Growth neutral medium growth Growth in NaCl 7% Slow growth Poor growth Good growth Growth in glu- Very slow Growth Growth cose-asparagine growth medium Voges-Proskauer Negative .+-. .+-. reaction Growth at 56.degree. C Growth Very poor Poor growth growth Membranes forma- No formation No forma- Formation tion tion ______________________________________
As shown in the above table, it is clear that the said microorganisms are not identical to each other in their characteristics.
Then, comparative examinations of the said Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76 according to the method of classification described in the said "Aerobic Spore-forming Bacteria" and "Bergey's Manual of Determinative Bacteriology" (1957) (from p. 612, et seq.) showed that the said microorganisms had some property similar to the known microorganisms belonging to the Bacillus sp., but were entirely different in characteristic properties, and there were no species among the known genus which agreed with the afore-mentioned properties. It was, therefore, concluded that they would be appropriate to establish new strains of the Bacillus sp. Since the said microorganisms are each an aerobic, spore-forming bacteria, it is clear that it should belong to the Bacillus genus. The highly characteristic property of the said microorganisms is that the growth is especially good in an alkaline medium, the optimal pH being 8 - 10.
Some examinations were made on known bacterial species in relation to Bacillus sp. No. 221, and Bacillus subtilis may be cited as a comparative organism. Bacillus subtilis is positive to the Vogel-Proskauer reaction, and has an optimal pH of 5 - 8 in a medium containing glucose. In contrast, Bacillus sp. No. 221 is negative to the Vogel-Proskauer reaction, and shows poor growth in a medium containing glucose at pH 7, the optimal being around pH 10. In these respects, Bacillus sp. No. 221 is clearly discriminated from Bacillus subtilis.
Bacillus brevis may also be cited as a comparative organism for each of Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76. However, Bacillus brevis has no ability to hydrolyze starch, produces gas in a nitrate medium under anaerobic conditions, and its gram stainability is varied, usually being negative. In contrast, Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76 have each a slight ability to hydrolyze starch, do not produce gas in a nitrate medium under anaerobic conditions, and are invariably positive in gram staining. The most characteristic difference in their properties is that whereas Bacillus brevis has pH 8.0 - 8.6 as its growth condition but does not grow at all at pH 10, Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76 grow at pH 7 - 11. Growth of each of Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76 is very slow at pH 7 in the presence of glucose, and the pH of around 10 is optimal. In this respect, each of Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76 is clearly distinguished from Bacillus brevis.
Since growth of Bacillus sp. No. 221 was inhibited by glucose, it may be compared with Bacillus firmus. The growth of these two microorganisms on citrate medium and the optimal temperature of their growth are different. In addition, Bacillus sp. No. 221 shows glucose inhibition at pH 7, but at pH 10 no growth inhibition by glucose is detected, however, Bacillus firmus does not grow at pH 10, irrespective of the presence or absence of glucose. In these respect, the two microorganisms are clearly distinguished.
Consequently, it is appropriate to establish a new species for each of the said microorganisms, Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76.
According to the process of the present invention, the alkaline protease of the present invention can be produced using not only Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76, described above, but also natural and artificial mutants thereof producing the said alkaline protease.
In the practice of the present invention, the fermentation may be carried out according to the following method.
First, there are two methods for the preparation of the culture medium used in the present invention.
The first method is characterized by the addition of carbonate to the composition of the culture medium. In this medium, glucose, starch, dextrin, maltose, fructose, and the like are used as the carbon source. Yeast extract, peptone, corn-steep liquor, and the like are used as the nitrogen source. Various carbonates, such as potassium carbonate, sodium carbonate, and sodium bicarbonate are used as the inorganic salts and added to the solution to prepare the culture medium which is adjusted to about pH 10.5.
The second method is characterized by the elimination of glucose and starch from the composition of the culture medium. In this case, a carbonate may be added to the culture medium as an inorganic salt. Yeast extract, peptone, corn-steep liquor, and the like are used as the nitrogen source, and various carbonates such as potassium carbonate, sodium carbonate, and sodium bicarbonate are used as the inorganic salts and may be added to the solution of the former to prepare the culture medium which is adjusted to about pH 7.
The medium thus prepared is inoculated with the strain of a microorganism selected from the group consisting of Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76, and cultured with shaking at a temperature of about 24.degree. - 45.degree. C. In general, the activity of the enzyme produced reaches a maximum after about 24 - 75 hours, e.g., 30 - 50 hours, of culture.
Since the period required to reach the maximum enzyme activity may vary according to the aeration and stirring conditions, even when the same temperature and culture medium of the same components are used, it is advisable to decide the period of culture by measuring the enzyme activity in each case.
The second important condition is the pH value of the medium. It is necessary to adjust the initial pH value within the range of 6 to 11 with carbonate or bicarbonate salts. Further, the optimal pH value when using a culture medium containing sugars is 8 to 11, while the pH of 6 to 10 is the optimal when glucose has been eliminated from the culture medium.
The commonly used physicochemical methods can be employed for the isolation of the enzyme from the culture broth. For example, after cooling the culture broth, acetic acid is added or not added to the culture broth to neutralize it, and ethanol is then added to precipitate the enzyme, alkaline protease, quantitatively. The precipitate is then collected, thoroughly washed with ethanol, and dried. The enzyme, alkaline protease, thus obtained from the culture of the said microorganisms is confirmed to be an alkaline protease having the optimum pH of about 11.5 and retaining the activity, even when heated at 50.degree. C for 1 hour.
As the culture condition, it is necessary to have an alkaline medium containing a high concentration of carbonate. Various carbonates are added to the medium containing a composition such as the carbon source and nitrogen source necessary fot the growth of the microorganism. For example, it is necessary to make a medium containing glucose, K.sub.2 HPO.sub.4, yeast extract, peptone, and MgSO.sub.4.7H.sub.2 O, with the addition of sodium carbonatee, potassium carbonate, or sodium bicarbonate. It is desirable to make the concentration of the carbonate added to 0.5 - 5%.
For the advantageous production of the objective alkaline protease, addition of a carbonate to the above medium is an extremely important condition and the following fact is proved from experimental results.
The growth of the microorganism used, Bacillus sp. No. 221, and production of protease were examined by the use of the foregoing medium with 1% of sodium carbonate or 1% each of various carbonates, and the same medium from which the carbonate was eliminated and in its place 1% of sodium chloride or potassium chloride, and adjusted to pH 10.0 with sodium hydroxide. Growth of the microorganism (said Bacillus sp. No. 221) was tested by taking the culture broth after 18 hours into a cuvette of 1 cm light path and measuring its absorbance at 660 m.mu., and protease activity was measured under the conditions described hereinbelow. These results are listed in Table 1, which indicates that the presence of a carbonate in the medium is an important factor for the production of the alkaline protease of the present invention.
Table 1 __________________________________________________________________________ Glucose Protease concen- Initial Final activity tration Salt added pH pH Growth (U/ml) __________________________________________________________________________ 1 None 7.2 6.5 0.1 &lt;100 (Only containing glu- cose, K.sub.2 HPO.sub.4, yeast extract, peptone, and MgSO.sub.4 . 7H.sub.2 O) 1 KCl 1% 10.5 -- 0.5 &lt;100 (adjusted with NaOH) 1 NaCl 1% 10.5 -- 0.45 &lt;100 (adjusted with NaOH) 1 Na.sub.2 HPO.sub.4 --NaOH 10.5 -- 0.8 750 0.05 = M 1 NaHCO.sub.3 1.0% 10.0 8.5 1.2 1400 1 Na.sub.2 CO.sub.3 0.5% 10.2 8.8 1.2 1800 1 Na.sub.2 CO.sub.3 1.0% 10.5 9.3 1.2 2800 1 K.sub.2 CO.sub.3 1.0% 10.5 9.2 1.15 1500 __________________________________________________________________________
Next, the growth of the microorganism used, Bacillus sp. No. 0-4, and production of protease were examined by the use of the foregoing medium with 1% of sodium carbonate or 1% each of various carbonates, and the same medium from which the carbonate was eliminated and in its place 1% of sodium chloride or potassium chloride, and adjusted to pH 10.0 with sodium hydroxide. Growth of the microorganism was tested by taking the culture broth after 18 hours into a cuvette of 1 cm light path and measuring its absorbance at 660 m.mu., and protease activity was measured under the condition described in later. These results are listed in Table 2, which indicates that the presence of a carbonate in the medium is an important factor for the production of the alkaline protease of the present invention.
In addition, when the said Bacillus sp. No. Y-76 was used as a test organism, results similar to those of Table 2 were obtained.
Table 2 ______________________________________ Protease activity Salt added pH Growth (U/ml) ______________________________________ None 10.0 0.8 750 (Only containing soluble starch, K.sub.2 HPO.sub.4, yeast extract, peptone and MgSO.sub.4 . 7H.sub.2 O) KCl " 0.9 " NaCl " " " Na.sub.2 CO.sub.3 10.5 1.2 71.00 NaHCO.sub.3 9.0 " 51.00 K.sub.2 CO.sub.3 10.5 1.1 52.00 ______________________________________
The other medium used in the present invention is a medium not containing any sugar. It is a medium containing components necessary for the growth of microorganism such as carbon source and nitrogen source, such as K.sub.2 HPO.sub.4, yeast extract, peptone, and MgSO.sub.4.7H.sub.2 O. It is desirable that the pH value be adjusted to 6 - 10.
For the advantageous production of the alkaline protease, the absence of sugars in the foregoing medium is an important condition and this fact is proved by the following experiments.
The growth of the organism used, Bacillus sp. No. 221, and the production of the alkaline protease were examined by the use of the said medium, to which was added sodium carbonate or sodium bicarbonate. The same medium with glucose, with or without sodium carbonate or sodium bicarbonate was tested. The growth of the organism (said Bacillus sp. No. 221) was examined by taking the culture broth after 18 hours in a cuvette of 1 cm light path and measuring its absorbance at 660 m.mu., and protease activity was measured under the condition described below. These results are given in Table 3 which indicates that the absence or low concentration of glucose in the medium is an important factor at neutral pH for the production of the alkaline protease of the present invention.
Table 3 __________________________________________________________________________ (When glucose is not added) Glucose Protease concentra- Salt (carbonate) Initial Final activity tion (%) concentration(%) pH pH Growth (U/ml) __________________________________________________________________________ 0 None (Only K.sub.2 HPO.sub.4, yeast 6.8 9.3 1.1 2500 extract, peptone, MgSO.sub.4.7H.sub.2 O) NaHCO.sub.3 0.1 8.2 9.4 1.0 2700 " 0.2 8.7 9.5 0.9 2000 " 0.3 9.1 9.6 1.0 1800 Na.sub.2 CO.sub.3 0.2 9.5 9.6 0.9 1600 " 1.0 10.6 9.8 0.8 1150 __________________________________________________________________________ (When glucose is added) 0.1 None 7.2 9.0 1.1 600 (Only glucose, K.sub.2 HPO.sub.4, yeast extract, pep- tone, MgSO.sub.4.7H.sub.2 O) NaHCO.sub.3 0.2 8.7 9.3 1.0 2600 " 0.5 9.6 9.4 1.0 2100 " 1.0 10.0 9.5 0.98 2100 Na.sub.2 CO.sub.3 0.2 9.3 9.3 0.98 1800 " 0.4 10.0 9.5 1.1 1600 "1.0 10.5 9.5 1.05 600 __________________________________________________________________________ 0.5 0 7.2 6.0 0.14 100 NaHCO.sub.3 0.2 8.6 6.4 0.40 600 " 0.5 9.6 9.3 1.2 1600 " 1.0 10.0 9.3 1.15 2000 Na.sub.2 CO.sub.3 0.2 9.2 9.1 1.1 1600 " 0.4 10.0 9.3 1.1 1100 " 1.0 10.5 9.6 1.15 900 __________________________________________________________________________ 1.0 Na.sub.2 CO.sub.3 1.0% 7 (adjusted 0.4 200 with HCl or NaOH) " 8 " 0.9 900 " 9 " 1.2 1500 " 10 " 1.2 2100 " 11 " 0.7 700 __________________________________________________________________________
The next important point in the culture condition is the pH during the culture. From the results of the following experiments, it is necessary to adjust the pH for the production of the alkaline protease to the selected value in the range of 6 to 11. Examination of the effect of pH on the production of alkaline protease by varying the pH of the above culture medium containing 1% of sodium carbonate with HCl or NaOH, as shown in Table 4, indicated that the best result was obtained at pH 7 - 11, especially at pH 8 - 11, in the presence of glucose. In this case, the microorganism used was the said Bacillus sp. No. 221.
Table 4 ______________________________________ pH Protease Activity (U/ml) ______________________________________ Medium 7 8 9 10 11 Containing 220 880 1,520 1,640 700 1% Na.sub.2 CO.sub.3 Not containing 100 100 100 100 100 1% Na.sub.2 CO.sub.3 ______________________________________
Next, when the said Bacillus sp. No. 0-4 was used, the results as shown in Table 5 were obtained.
Table 5 ______________________________________ pH Protease Activity (U/ml) ______________________________________ Medium 6.8 8 9 10 11 Containing 800 3,000 5.550 6,300 2,530 1% Na.sub.2 CO.sub.3 ______________________________________
From the Table 5, it is noted that the best result is obtained at pH 6 - 11, especially at pH 8 - 11, in the presence of carbonate.
In addition, when the said Bacillus sp. Y-76 was used as a test organism results similar to those of Table 5 were obtained.
The effect of pH on the production of alkaline protease was then examined by varying the pH of the culture medium with NaHCO.sub.3 or Na.sub.2 CO.sub.3 and not containing sugars, and the result is shown in Table 6. When glucose was not added, the best result was obtained in a pH range of 6 - 11, especially at 6 - 10. The microorganism used was the said Bacillus sp. No. 221.
Table 6 ______________________________________ Protease Initial Final activity Salt added pH pH Growth (U/ml) ______________________________________ None (only K.sub.2 HPO.sub.4, yeast 7.0 9.3 1.2 2,100 extract, peptone, MgSO.sub.4.7H.sub.2 O) Na.sub.2 CO.sub.3 0.5% 6.8* 9.2 1.1 2,000 NaHCO.sub.3 0.1% 8.2 9.4 1.0 1,800 " 0.2% 8.7 9.5 0.9 1,580 " 0.3% 9.1 9.6 1.0 1,300 Na.sub.2 CO.sub.3 0.2% 9.5 9.6 0.9 1,020 " 1.0% 10.3 9.8 0.8 420 ______________________________________ *adjusted with HCl
Thus, under the foregoing culture conditions, each of the said microorganisms, Bacillus sp. No. 221, Bacillus sp. No. 0-4 and Bacillus sp. No. Y-76, preincubated in the same medium, is inoculated in the medium, and this is shakecultured under appropriate condition, such as 24 - 75 hours at 37.degree. C. After completion of the culture, cells are removed, the broth is neutralized with acetic acid or a similar acid, or not neutralized, and then an organic solvent like ethanol or acetone is added to precipitate the alkaline protease produced. The precipitate is then dehydrated and dried to obtain the objective substance.
The activity of the alkaline protease so obtained is ca. 3,000 U/ml, the said alkaline protease has an optimal pH in the alkaline side of around 11.5. This alkaline protease does not lose its activity even when heated at 50.degree. C for 1 hour, proved by the result of experiments.
Physicochemical properties of the enzyme of this invention, alkaline protease, are as follows:
The crystalline enzyme of the present invention, with buffer solution of various pH's, was heated at 60.degree. C for 10 minutes, and the residual activity was determined. Its result is shown in FIG. 5.
______________________________________ Buffer solutions used: pH ______________________________________ Acetate buffer 4 - 6 Phosphate buffer 6 - 8 All in 0.05 =M NaHCO.sub.3 -- Na.sub.2 CO.sub.3 buffer 9 - 10.7 Na.sub.2 HPO.sub.4 -- NaOH buffer 11 - 12 ______________________________________
One milliliter of the enzyme solution suitably diluted with 10.sup.-2 M CaCl.sub.2 was mixed with 5 ml of 0.6% casein solution (pH 11.5, 2 .times. 10.sup.-2 M Na.sub.2 HPO.sub.4 - NaOH buffer) at 30.degree. C. After 10 minutes' incubation, 5 ml of trichloroacetic acid solution (0.11M trichloroacetic acid, 0.22M sodium acetate, and 0.33M acetic acid) was added to the reaction mixture and the mixture was further incubated at 30.degree. C for 30 minutes. The mixture was filtered and the absorbance of the filtrate was measured at 275 m.mu.. The readings were corrected by substracting the value of the blank in which the enxyme solution was mixed with trichloroacetic acid solution before the casein solution was added.